Blood vessel model

ABSTRACT

The present disclosure provides a blood vessel model including: a pair of channel members, mutually opposing each other, each of which includes an opposing face in which a respective microchannel is formed; and a porous membrane that includes plural through-holes penetrating in a thickness direction, that is disposed between the opposing faces of the pair of channel members, and that partitions between the microchannels, wherein the porous membrane is provided with a vascular endothelial cell layer so as to cover one face facing one of the microchannels, an average opening diameter of the through-holes is from 1 μm to 20 μm, and an opening coverage ratio of the through-holes is from 30% to 70%.

BACKGROUND Technical Field

The present disclosure relates to a blood vessel model.

Related Art

Recently, there have been attempts to model internal organs such asblood vessels, intestines, livers, and lungs, using devices includingwhat are referred to as microchannels, which include channels withmicrometer order widths. United States Patent Application Publication(US) No. 2011/0053207, Japanese Patent Application Publication (JP-B)No. 5415538, and JP-B No. 5815643, for example, each discloses aninternal organ model including a porous membrane having a layer of cellsprovided on a surface thereof, and at least two microchannelspartitioned by the porous membrane.

Various experiments and tests can be performed using an internal organmodel such as that disclosed in US 2011/0053207, JP-B No. 5415538, andJP-B No. 5815643. For example, what is called an extravasation test maybe performed by running blood containing a drug through one of themicrochannels, and then measuring the number or amount of red bloodcells, biomarker, or the like, which have moved through the porousmembrane from the one microchannel to another microchannel. Thisextravasation test enables evaluation of the level of drug inducedinjury to the layer of cells provided to the surface of the porousmembrane, enabling drug toxicology testing to be performed.

However, the pores in the porous membrane used in conventional internalorgan models are produced using what is known as a track etchingprocess, in which, for example, a material configuring the porousmembrane is irradiated with heavy ions. Accordingly, the openingcoverage ratio of pores in the membrane is, for example, as low as from2% to 20%, and due to the membrane also being thick, the passage of redblood cells or the like is obstructed by the porous membrane. Namely, inthe conventional internal organ models, there were cases in which thelevel of drug induced injury to a layer of cells provided at a surfaceof the porous membrane may not be accurately evaluated.

SUMMARY

The present disclosure provides a blood vessel model that may enablemovement of red blood cells or the like to be suppressed from beingobstructed by a porous membrane during an extravasation test.

A blood vessel model according to a first aspect of the presentdisclosure includes: a pair of channel members, mutually opposing eachother, each of which includes an opposing face in which a respectivemicrochannel is formed; and a porous membrane that includes pluralthrough-holes penetrating in a thickness direction, that is disposedbetween the opposing faces of the pair of channel members, and thatpartitions between the microchannels, wherein the porous membrane isprovided with a vascular endothelial cell layer so as to cover one facefacing one of the microchannels, an average opening diameter of thethrough-holes is from 1 μm to 20 μm, and an opening coverage ratio ofthe through-holes is from 30% to 70%.

In the above configuration, the average opening diameter of thethrough-holes in the porous membrane partitioning between themicrochannels is from 1 μm to 20 μm, and the opening coverage ratio ofthe through-holes is from 30% to 70%. Thus, during extravasationtesting, when red blood cells or the like that are flowing through thethrough-holes in the porous membrane and moving from one of themicrochannels to the other of the microchannels, the movement of the redblood cells or the like may be suppressed from being obstructed by theporous membrane.

In a second aspect of the present disclosure, in the first aspect, amembrane thickness of the porous membrane may be less than or equal tohalf of the average opening diameter of the through-holes.

In the above second aspect, since the membrane thickness of the porousmembrane is less than or equal to half of the average opening diameterof the through-holes, compared to a case in which the membrane thicknessof the porous membrane is greater than half the average opening diameterof the openings of the through-holes, red blood cells or the like mayfurther readily pass through the through-holes in the porous membrane.Accordingly, the second aspect may further improve the accuracy of theextravasation test.

In a third aspect of the present disclosure, in the first or secondaspect, communication holes that place the through-holes incommunication with each other may be formed inside the porous membrane;the through-holes may be arranged in a honeycomb pattern; a variationcoefficient of opening diameters of the through-holes may be less thanor equal to 10%; and a porosity of the porous membrane may be greaterthan or equal to 50%.

In the above third aspect, the through-holes are arranged in a honeycombpattern and are in communication with each other through thecommunication holes. The variation coefficient of the opening diametersof the openings of the through-holes is less than or equal to 10%, andthe porosity of the porous membrane is greater than or equal to 50%.Thereby, in the third aspect, the red blood cells or the like may bemade to pass through more uniformly. Accordingly, the third aspect mayfurther improve the accuracy of the extravasation test.

In a fourth aspect of the present disclosure, in the first to the thirdaspects, a cell layer of cells may be selected from the group consistingof smooth muscle cells, mesenchymal stem cells, pericytes, andfibroblast cells, and may be provided at the other face of the porousmembrane facing the other microchannel.

In the above fourth aspect, due to forming the cell layer of cells fromthe group consisting of smooth muscle cells, mesenchymal stem cells,pericytes, and fibroblast cells, on the other face of the porousmembrane on the opposite side to the face on which the vascularendothelial cell layer is formed, a blood vessel model that more closelyresembles an actual blood vessel may be achieved.

In a fifth aspect of the present disclosure, in the first aspect to thefourth aspect, a tensile elongation at break of the porous membrane maybe greater than or equal to 50%; and a stress required for 10%elongation of the porous membrane may be less than or equal to 1000gf/mm².

In the above fifth aspect, since the porous membrane is formed from aflexible material having a tensile elongation at break greater than orequal to 50% and having a stress required for 10% elongation less thanor equal to 1000 gf/mm², a blood vessel model that more closelyresembles an actual blood vessel may be achieved.

In a sixth aspect of the present disclosure, in the first aspect to thefifth aspect, the through-holes may have flattened shapes in plan viewand may include a major axis and a minor axis.

In the above sixth aspect, since the through-holes have flattened shapessuch as elliptical shapes in plan view, the red blood cells or the likemay pass more readily through the through-holes. Accordingly, the sixthaspect may further improve accuracy of the extravasation test.

In a seventh aspect of the present disclosure, in the first aspect tothe sixth aspect, the porous membrane may include a porous region inwhich the through-holes are formed, and a non-porous region in which thethrough-holes are not formed.

In the above seventh aspect, since, for example, portions of the porousmembrane disposed in the vicinity of inlets and in the vicinity ofoutlets of the microchannels are configured as the non-porous regions inwhich the through-holes are not formed, the flow of red blood cells orthe like inside the microchannels may be regulated. Accordingly, theseventh aspect may further improve accuracy of the extravasation test.

According to the above aspects, the present disclosure may enable themovement of red blood cells or the like during extravasation testing tobe suppressed from being obstructed by the porous membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the present invention will be described indetail based on the following figures, wherein:

FIG. 1 is a perspective diagram illustrating an overall configuration ofa blood vessel model of an exemplary embodiment;

FIG. 2 is an exploded perspective diagram illustrating overallconfiguration of a blood vessel model of an exemplary embodiment;

FIG. 3 is an enlarged cross-section illustrating a porous membrane of ablood vessel model of an exemplary embodiment;

FIG. 4 is a plan view illustrating a porous membrane of a blood vesselmodel of an exemplary embodiment;

FIG. 5 is a plan view illustrating a porous membrane of a blood vesselmodel of a modified example;

FIG. 6 is a plan view illustrating a porous membrane of a blood vesselmodel of a modified example;

FIG. 7A is a micrograph of a porous membrane of an example 1; and

FIG. 7B is a micrograph of a porous membrane of a comparative example 1.

DETAILED DESCRIPTION

Explanation follows regarding an example and modified examples of anexemplary embodiment of the present disclosure, with reference to FIG. 1to FIG. 6. Note that the following exemplary embodiment is merely anexample of the present disclosure, and does not limit the scope of thepresent disclosure. Also note that the dimensions of variousconfiguration in the drawings are modified as appropriate in order tofacilitate explanation of the various configuration. Accordingly, thescale in the drawings may differ from the scale in actual practice.

As illustrated in FIG. 1 and FIG. 2, a blood vessel model 10 of anexemplary embodiment includes an upper channel member 12 and a lowerchannel member 14 stacked on one another. The upper channel member 12and the lower channel member 14 are, for example, configured from anelastic material such as polydimethylsiloxane (PDMS), and havesubstantially rectangular plate shapes.

A concave portion 18, that defines an upper microchannel 16, is formedin a lower face of the upper channel member 12, namely, at an opposingface 12A opposing to the lower channel member 14. The concave portion 18includes an inlet 18A, an outlet 18B, and a channel portion 18Ccommunicating the inlet 18A with the outlet 18B.

Through-holes 20A and 20B are formed in the upper channel member 12,penetrate the upper channel member 12 in the thickness direction, andhave lower ends in respective communication with the inlet 18A and theoutlet 18B. Upper ends of the through-holes 20A and 20B open at an upperface of the upper channel member 12. Liquid supply tubing (notillustrated) is connected to the upper ends of the through-holes 20A and20B.

Similarly, a concave portion 24 that defines a lower microchannel 22 isformed in an upper face of the lower channel member 14, namely, at anopposing face 14A opposing to the upper channel member 12. The concaveportion 24 includes an inlet 24A, an outlet 24B, and a channel portion24C communicating the inlet 24A with the outlet 24B.

The inlet 24A and outlet 24B of the lower channel member 14 and theinlet 18A and outlet 18B of the upper channel member 12 are provided atpositions not overlapping in plan view. In contrast thereto, the channelportion 24C of the lower channel member 14 and the channel portion 18Cof the upper channel member 12 are provided at positions overlapping inplan view.

Through-holes 26A and 26B are also formed in the upper channel member12, penetrate the upper channel member 12 in the thickness direction,and have lower ends in respective communication with the inlet 24A andthe outlet 24B. Upper ends of the through-holes 26A and 26B open at theupper face of the upper channel member 12. Liquid supply tubing (notillustrated) is connected to the upper ends of the through-holes 26A and26B.

A porous membrane 28 is provided between the opposing faces 12A and 14Aof the upper channel member 12 and the lower channel member 14. Theupper channel member 12 and the lower channel member 14 are joinedtogether with the porous membrane 28 in an interposed statetherebetween. Note that, besides being bonded together using an adhesiveagent, a variety of methods, such as welding, attraction(self-adhering), or joining with bolts, may be employed as the methodfor joining the upper channel member 12 and the lower channel member 14together.

The porous membrane 28 is, for example, a hydrophobic polymer thatsolves in a hydrophobic organic solvent. Note that the hydrophobicorganic solvent is a liquid with a solubility in 25° C. water of lessthan or equal to 10 (g/100 g water).

Examples of hydrophobic polymers include polymers such as polybutadiene,polystyrene, polycarbonate, polyesters (for example, polylactic acid,polycaprolactone, polyglycolic acid, polylactic acid-polyglycolic acidcopolymer, polylactic acid-polycaprolactone coolymer, polyethyleneterephthalate, polyethylene naphthalate, polyethylene succinate,polybutylene succinate, and poly-3-hydroxybutyrate), polyacrylate,polymethacrylate, polyacrylamide, polymethacrylamide, polyvinylchloride, polyvinylidene chloride, polyvinylidene fluoride,polyhexafluoropropene, polyvinyl ether, polyvinylcarbazole, polyvinylacetate, polytetrafluoroethylene, polylactone, polyamide, polyimide,polyurethane, polyurea, polyaromatics, polysulfone, polyethersulfone,polysiloxane derivatives, and cellulose acylate (for example, triacethylcellulose, cellulose acetate propionate, and cellulose acetatebutyrate). Polymers that dissolve in a hydrophobic organic solvent arepreferable from the viewpoint of producing a honeycomb membrane usingthe production method disclosed, for example, in Japanese Patent No.4,734,157.

These polymers may have the form of a homopolymer, a copolymer, apolymer blend or a polymer alloy, as necessary, from the viewpoints of,for example, solubility in solvents, optical properties, electricalproperties, membrane strength, and elasticity. These polymers may beused singly, or in combination of two or more thereof. Note that thematerial of the porous membrane 28 is not limited to being a hydrophobicpolymer, and various materials may be selected from viewpoints such asthe adhesiveness of cells.

An upper face 28A and a lower face 28B of the porous membrane 28(hereinafter, the upper face 28A and the lower face 28B are may bereferred collectively as “main faces”) are sized so as to substantiallycover the channel portions 18C and 24C of the upper microchannel 16 andthe lower microchannel 22, such that the upper microchannel 16 ispartitioned from the lower microchannel 22.

Specifically, the upper face 28A of the porous membrane 28, namely, themain face facing the upper channel member 12, together with the concaveportion 18 of the upper channel member 12, defines the uppermicrochannel 16. The lower face 28B of the porous membrane 28, namely,the main face facing the lower channel member 14, together with theconcave portion 24 of the lower channel member 14, defines the lowermicrochannel 22.

As illustrated in FIG. 3, a vascular endothelial cell layer 36, forexample, is provided to the upper face 28A of the porous membrane 28 soas to completely cover the upper face 28A. The inside of the uppermicrochannel 16 thereby configures an environment that closely resemblesthe inside of a blood vessel. Examples of vascular endothelial cellsinclude: vascular endothelial cells originating from the umbilical vein,the umbilical artery, the aorta, a coronary artery, the pulmonaryartery, a pulmonary microvessel, or a dermal microvascular; and vascularendothelial cells differentiated from pluripotent stem cells.

A cell layer 38, for example, configured from cells selected from thegroup consisting of smooth muscle cells, mesenchymal stem cells,pericytes, and fibroblast cells, is provided to the lower face 28B ofthe porous membrane 28 so as to completely cover the lower face 28B. Thelower microchannel 22 thereby configures an environment that closelyresembles a blood vessel exterior. Mesenchymal stem cells (MSC) aresomatic stem cells that are capable of dividing into muscle cells, fatcells, cartilage cells, and the like.

Note that the cell layer 38 of cells selected from the group consistingof smooth muscle cells, mesenchymal stem cells, pericytes, andfibroblast cells may be provided to the upper face 28A of the porousmembrane 28, and the vascular endothelial cell layer 36 may be providedto the lower face 28B of the porous membrane 28. Moreover, it issufficient that the vascular endothelial cell layer 36 be provided to atleast one of the main faces of the porous membrane 28. Configuration maybe such that the cell layer 38 is not provided to the other main face ofthe porous membrane 28.

From the viewpoint of adhesiveness of cells, it is preferable that aregion where cells are seeded on at least one of the upper face 28A andthe lower face 28B of the porous membrane 28 is coated by at least oneselected from the group consisting of fibronectin, collagen (forexample, type I collagen, type IV collagen or type V collagen), laminin,vitronectin, gelatin, perlecan, nidogen, proteoglycan, osteopontin,tenascin, nephronectin, a basement membrane matrix and polylysine. Notethat it is preferable that the porous membrane 28, and the inside ofthrough-holes 30, described later, be coated by at least one of these.

For providing the vascular endothelial cell layer 36 and the cell layer38 to the respective main face of the porous membrane 28, for example, amethod in which a cell suspension is poured into the upper microchannel16 and the lower microchannel 22 so as to seed cells on the main facesof the porous membrane 28, may be employed. Further, a method in whichcells are seeded and cultured on the main faces of the porous membrane28 inside a separate culturing apparatus, and then the porous membrane28 having the vascular endothelial cell layer 36 and the cell layer 38formed thereon is mounted in the blood vessel model 10, may also beemployed.

As illustrated in FIG. 3 and FIG. 4, plural through-holes 30 are formedin the porous membrane 28 penetrating the porous membrane 28 in thethickness direction. Openings 30A of the through-holes 30 are providedin each of the upper face 28A and the lower face 28B of the porousmembrane 28. As illustrated in FIG. 4, the openings 30A have circularshapes in plan view. The openings 30A are provided at a separation fromeach other. A flat portion 32 extends between adjacent openings 30A.Note that the openings 30A are not limited to circular shapes, and mayalso be configured with polygonal shapes.

The plural openings 30A are arranged in a regular manner, and in thepresent exemplary embodiment, as illustrated in FIG. 4, for example, arearranged in honeycomb pattern. A honeycomb pattern arrangement is anarrangement in which the centers of the openings 30A are disposed atpositions of the vertexes, and at the points where diagonals intersect,for units of a parallel hexagon (a regular hexagon is preferable) or ashape close to this. Herein, “centers of the openings” means the centersof the openings 30A in plan view.

Note that the arrangement of the openings 30A is not limited to ahoneycomb pattern. The openings 30A may also be configured in a latticepattern or a face-centered lattice pattern. A lattice patternarrangement is an arrangement in which the centers of the openings aredisposed at the positions of the vertexes for units of a parallelogram(it goes without saying that this includes squares, rectangles, andrhombuses; a square is preferable) or a shape close thereto. Aface-centered lattice pattern arrangement is an arrangement in which thecenters of the openings are disposed at the positions of the vertexes,and at the points where diagonals intersect, for units of aparallelogram (it goes without saying that this includes squares,rectangles, and rhombuses; a square is preferable) or a shape closethereto.

The arrangement of the openings 30A may be arbitrary. However, it ispreferable that the plural openings 30A are arranged in a regular mannerfrom the viewpoint of achieving a uniform density of the openings 30A inthe upper face 28A and the lower face 28B of the porous membrane 28. Aregular arrangement is an arrangement in which the variation coefficientof the surface areas of the parallel hexagon or parallelogram units ofthe arrangement is, for example, less than or equal to 10%. Some of theopenings 30A may be missing or the openings 30A may be not in alignment.However, it is preferable that the openings 30A are continuouslyarranged in all directions without any gaps therebetween. Note that the“variation coefficient” is a value arrived at by dividing the standarddeviation of a given population by the mean thereof, and is an indexexpressing the degree of dispersion in the population as a percentage.

As illustrated in FIG. 3, each through-hole 30 in the porous membrane 28has a spherical segment shape, which is a shape in which the upper andlower end of a sphere have been cut away. Through-holes 30 that areadjacent to one another are in communication with each other throughrespective communication holes 34 in the interior of the porous membrane28.

It is preferable that each through-hole 30 is in communication withevery adjacent through-hole 30. In cases in which the openings 30A ofthe plural through-holes 30 are arranged in a honeycomb pattern, as inthe present exemplary embodiment, each through-hole 30 is in respectivecommunication with six adjacent through-holes 30 through sixcommunication holes 34. Note that the through-holes 30 may have a barrelshape, a circular column shape, a polygonal column shape, or the like,and the communication holes 34 may be tube shaped voids linking togetheradjacent through-holes 30.

An opening diameter D of each opening 30A of the through-holes 30 is,for example, a size such that red blood cells in blood are able to passtherethrough. Specifically, the average opening diameter is preferablyfrom 1 μm to 20 μm, and even more preferably is from 3 μm to 10 μm.Setting the average opening diameter to 1 μm or greater enables thethrough-holes 30 to be a size that allows red blood cells to passtherethrough, and setting the average opening diameter to 20 μm or lessenables retention of the vascular endothelial cell layer 36 and the celllayer 38 on the main faces of the porous membrane 28.

Herein, “opening diameter D” is the major axis of the openings 30A, and“average opening diameter” is the calculated average of the openingdiameters D measured for ten or more arbitrarily selected openings 30A.“Major axis” means the longest distance between two arbitrarily selectedpoints on the outline of an opening. However, in cases in which adirection has been specified, “major axis” means the longest distancebetween two arbitrarily selected points along that direction.

The opening coverage ratio of the openings 30A of the through-holes 30is preferably from 30% to 70%, and even more preferably is from 40% to60%. Setting the opening coverage ratio to greater than or equal to 30%enables the movement of red blood cell to be suppressed from beingobstructed by the porous membrane 28, and setting the opening coverageratio to less than or equal to 70% enables the required strength to beachieved in the porous membrane 28.

Herein, “opening coverage ratio” expresses a ratio of S2 to S1 as apercentage, wherein S1 denotes a unit of surface area of the porousmembrane 28 under the supposition that the main faces of the porousmembrane 28 are smooth (namely, under the supposition that there are noopenings 30A in the porous membrane 28), and S2 denotes the sum of thesurface area of openings 30A provided per unit surface area, where thesame units of measurement are used for S1 and S2.

The membrane thickness T of the porous membrane 28 is preferably lessthan or equal to half of the average opening diameter of the openings30A of the through-holes 30. Specifically, the thickness T is preferablyfrom 0.5 μm to 10 μm, and is more preferably from 1 μm to 10 μm. Settingthe membrane thickness T of the porous membrane 28 to a thickness lessthan or equal to half of the average opening diameter of thethrough-holes 30 enables the movement of red blood cell to be suppressedfrom being obstructed by the porous membrane 28.

The variation coefficient of the opening diameters D of the openings 30Ais preferably less than or equal to 10%, and the smaller the variationcoefficient the more preferable. The smaller the variation coefficientof the opening diameters D, the more uniformly red blood cells and thelike can pass through the plural through-holes 30 in the porous membrane28.

The porosity of the porous membrane 28 is preferably greater than orequal to 50%. Setting the porosity to greater than or equal to 50%enables the movement of red blood cell to be suppressed from beingobstructed by the porous membrane 28. Note that if the porosity is toolarge, the strength of the porous membrane 28 becomes insufficient withregards to the required strength therefor, and so the porosity ispreferably less than or equal to 95%.

Herein, “porosity” expresses a ratio of V2 to V1 as a percentage,wherein V1 denotes a unit of volume of the porous membrane 28 under thesupposition that the main faces of the porous membrane 28 are smooth(namely, under the supposition that there are no openings 30A in theporous membrane 28), and V2 denotes the sum of the volume of thethrough-holes 30 and the communication holes 34 provided per unitvolume, where the same units of measurement are used for V1 and V2.

The tensile elongation at break of the porous membrane 28 is preferablygreater than or equal to 50%, is more preferably 100%, and is even morepreferably greater than or equal to 200%. The stress required for 10%elongation of the porous membrane 28 is preferably less than or equal to1000 gf/mm². A material becomes more flexible as the tensile elongationat break increases and the stress required for 10% elongation decreases.It is thus possible to bend, stretch, and compress the porous membrane28, enabling the blood vessel model 10 to more closely resemble anactual blood vessel.

Herein, the “tensile elongation at break” can be evaluated by measuringthe elongation at tensile breaking of the porous membrane 28 accordingto the method defined in JIS K 6251:2010. The “stress required for 10%elongation” can be evaluated by measuring the stress applied to theporous membrane 28 when the porous membrane 28 is elongated by 10%according to the method defined in JIS K 6251:2010.

Note that, examples of methods for producing the porous membrane 28formed with the through-holes 30 include, nano-printing processes,condensation processes, etching processes, sandblasting processes, orpress molding processes. A nano-printing process is a method in whichthe through-holes 30 are produced by pouring a material for configuringthe porous membrane 28 into a mold having projections and recesses, orpressing such a mold against a material for configuring the porousmembrane 28. A condensation process is a method in which condensation isinduced on the surface of a material for configuring the porous membrane28, so as to form the through-holes 30 by using water droplets as molds.

In comparison to other methods, a condensation process enables themembrane thickness of the porous membrane 28 to be made thinner, enablesthe porosity and the opening coverage ratio of the openings 30A to beincreased, and also enables the communication holes 34 to be providedwithin the porous membrane 28. Thus, in the present exemplaryembodiment, the porous membrane 28 is produced using a condensationprocess. Condensation processes are described in detail in, for example,JP-B No. 4945281, JP-B No. 5422230, JP-B No. 5405374, and JapanesePatent Application Laid-Open (JP-A) No. 2011-74140.

Next, as an example, explanation is given regarding a case in which adrug toxicology evaluation is performed using the blood vessel model 10of the present exemplary embodiment. When performing a drug toxicologytest, first, the upper channel member 12 and the lower channel member 14are joined together with the porous membrane 28 in an interposed statetherebetween to produce the blood vessel model 10 including the uppermicrochannel 16 and the lower microchannel 22, as illustrated in FIG. 2.Note that the vascular endothelial cell layer 36 and the cell layer 38are provided to the main faces of the porous membrane 28.

Then, using a pump, a blood dilution containing a drug is run throughtubing (not illustrated in the drawings) and the through-hole 20A intothe upper microchannel 16, is passed through the inside of the uppermicrochannel 16, and is caused to pass through the through-hole 20B andtubing (not illustrated in the drawings) to run out of the blood vesselmodel 10.

Meanwhile, using a pump, a culture solution or a physiological salinesolution is run through tubing (not illustrated in the drawings) and thethrough-hole 26A into the lower microchannel 22, is passed through theinside of the lower microchannel 22, and is caused to pass through thethrough-hole 26B and tubing (not illustrated in the drawings) to run outof the blood vessel model 10. Note that the pressure in the uppermicrochannel 16 through which the blood dilution flows is higher thanthat in the lower microchannel 22 through which the culture solution orthe physiological saline solution flows.

At the start of the toxicology test, as illustrated in FIG. 3, the wholeof the upper face 28A and the whole of the lower face 28B of the porousmembrane 28 are respectively covered by the vascular endothelial celllayer 36 and the cell layer 38. Accordingly, red blood cells in theblood are unable to pass through the porous membrane 28 and do not leakout into the lower microchannel 22.

However, when a certain amount of time has elapsed from the start of thetoxicology test, the vascular endothelial cell layer 36 is injured bythe toxicity of the drug. In addition to the vascular endothelial celllayer, the cell layer 38 is also injured by the drug. By measuring thenumber of red blood cells that have passed through the porous membrane28 and flowed into the lower microchannel 22 due to such an injuredportion, namely, by performing an extravasation test, it is possible toevaluate the level of drug induced injury to the vascular endothelialcell layer 36 and the cell layer 38.

The present exemplary embodiment is configured such that in the porousmembrane 28 partitioning the upper microchannel 16 from the lowermicrochannel 22, the average opening diameter of the openings 30A of thethrough-holes 30 is from 1 μm to 20 μm, and the opening coverage ratioof the openings 30A of the through-holes 30 is from 30% to 70%.Accordingly, during extravasation testing when red blood cells runningthrough the upper microchannel 16 pass through the through-holes 30 inthe porous membrane 28 and move into the lower microchannel 22, themovement of red blood cells can be suppressed from being obstructed bythe porous membrane 28.

Moreover, the present exemplary embodiment is configured such that themembrane thickness of the porous membrane 28 is less than or equal tohalf of the average opening diameter of the openings 30A of thethrough-holes 30. Accordingly, compared to a case in which the membranethickness of the porous membrane 28 is greater than half the averageopening diameter of the openings 30A of the through-holes 30, red bloodcells more readily pass through the through-holes 30 in the porousmembrane 28 Accordingly, the present exemplary embodiment may furtherimprove the accuracy of the extravasation test.

Additionally, the present exemplary embodiment is configured with theopenings 30A of the through-holes 30 arranged in a honeycomb pattern,and through-holes 30 within the porous membrane 28 are in communicationwith each other through the communication holes 34. The variationcoefficient of the opening diameters of the openings 30A of thethrough-holes 30 is less than or equal to 10%, and the porosity of theporous membrane 28 is greater than or equal to 50%. Accordingly, redblood cells can pass through the plural through-holes 30 in the porousmembrane 28 more uniformly. Accordingly, the present exemplaryembodiment may further improve the accuracy of the extravasation test.

Additionally, the present exemplary embodiment is configured with thevascular endothelial cell layer 36 provided to the upper face 28A of theporous membrane 28, and with the cell layer 38 provided to the lowerface 28B of the porous membrane 28. The cell layer 38 is configured ofcells selected from the group consisting of smooth muscle cells,mesenchymal stem cells, pericytes, and fibroblast cells. Additionally,the porous membrane 28 is configured from a flexible material having atensile elongation at break that is greater than or equal to 50%, and inwhich the stress required for 10% elongation is less than or equal to1000 gf/mm². Thereby, in the present exemplary embodiment, the bloodvessel model 10 may be configured to more closely resemble an actualblood vessel.

Explanation has been given regarding an example of an exemplaryembodiment of the present disclosure. However, the present disclosure isnot limited to the above, and various modifications may be implementedbesides the above, within a range not departing from the spirit of thepresent disclosure.

For example, although the openings 30A of the through-holes 30 in theporous membrane 28 of the above exemplary embodiment have circularshapes in plan view, openings 50A of through-holes 50 in a porousmembrane 48 may have elliptical shapes in plan view, as illustrated inFIG. 5. By configuring the openings 50A of the through-holes 50 withelliptical shapes, for example, disc-shaped red blood cells may beeasily passed through the openings 50A of the through-holes 50, andother cells in blood may be less liable to pass therethrough.

Examples of methods for forming the openings 50A of the openings 50A ofthrough-holes 50 into elliptical shapes include a method in which, aftercircular shaped openings 30A such as illustrated in FIG. 4 have beenformed in the porous membrane 48, the porous membrane 48 is stretchedalong one direction (the left-right direction in FIG. 4). This methodenables plural elliptical shaped openings 50A to be formed having theirmajor axis directions along the same direction (the left-right directionin FIG. 5).

Note that elliptical shaped openings 50A may be directly formed in theporous membrane 48 using press molding or the like, without stretchingthe porous membrane 48. Moreover, so long as the shape of the openings50A is a flattened shape with a major axis and a minor axis in planview, the shape of the openings 50A may, for example, be a flattenedpolygonal shape arrived at by stretching a regular polygon.

In the porous membrane 28 of the above exemplary embodiment, theopenings 30A of the through-holes 30 were arranged in a regular mannerover the entirety of the main faces of the porous membrane 28. However,as illustrated in FIG. 6, a porous membrane 58 may be provided with aporous region 62 in which openings 60A of through-holes 60 are formed,and a non-porous region 64 not formed with the openings 60A ofthrough-holes 60 (the region marked by the double-dotted dashed line inFIG. 6).

Specifically, in the porous membrane 58, portions disposed in thevicinity of the inlet 18A and in the vicinity of the outlet 18B of theconcave portion 18 configuring the upper microchannel 16 illustrated inFIG. 1, and in the vicinity of the inlet 24A and in the vicinity of theoutlet 24B of the concave portion 24 configuring the lower microchannel22 illustrated in FIG. 1, are, for example, configured as the non-porousregion 64.

Generally, the flow of liquid such as blood is easily disturbed at theinlets 18A and 24A and at the outlets 18B and 24B. Thus, by configuringthe porous membrane 58 in the vicinity of the inlets 18A and 24A and inthe vicinity of the outlets 18B and 24B as the non-porous region 64, theflow of liquid such as blood in the upper microchannel 16 and in thelower microchannel 22 may be regulated. Accordingly, the porous membrane58 may further improve the accuracy of the extravasation test.

The blood vessel model of the present disclosure may enableextravasation testing to be performed, in a state in which movement ofleaking substances such as red blood cells to outside the blood vesselaccompanying drug toxicity is suppressed from being obstructed by theporous membrane. The blood vessel model of the present disclosure maytherefore be useful as a blood vessel model capable of toxicologytesting with high-accuracy.

Detailed explanation follows regarding examples of the exemplaryembodiments of the present disclosure. Note that the exemplaryembodiments of the present disclosure are not to be interpreted as beinglimited by the examples illustrated below.

FIG. 7A illustrates a micrograph of a porous membrane of example 1. Inexample 1, a porous membrane, similar to the porous membrane 28 of theabove exemplary embodiment, was employed in which the openings of pluralthrough-holes were arranged in a honeycomb pattern and the through-holeswere in communication through communication holes. Note that the averageopening diameter of openings in the porous membrane of example 1 was 5the opening coverage ratio of the openings was 55%, the membranethickness of the porous membrane was 2.2 the variation coefficient ofthe opening diameters of the openings was 3.5%, the porosity of theporous membrane was 75%, the tensile elongation at break was 250%, andthe stress required for 10% elongation was 100 gf/mm2.

The microstructure of the produced porous membrane was measured using aprofile scanning laser microscope (product name VK-X100, made byKeyence, Japan). Observations were made using a magnification at which50 or more openings appeared on a single screen. Based on the observedmicrograph, image analysis was performed on the openings present on theone screen, so as to measure the respective opening diameters D and findthe average opening diameter DAV and the variation coefficient σD of theopening diameters D. Note that the variation coefficient of the openingdiameters (given as a percentage) can be achieved using the calculation(σD/DAV)×100.

The average opening diameter and the opening coverage ratio wereachieved by performing binarization processing and image processing onthe micrograph, using the 2D image analysis software WinROOF (MitaniCorp.). The membrane thickness of the porous membrane is an averagevalue of the thickness of opening portions measured at ten points usingthe profile scanning laser microscope.

Cross-sections of the porous membrane were observed using a scanningelectron microscope (SEM, product name SU8030, made by Hitachi, Japan)and the diameters of spheres equivalent to the through-holes wascalculated as the porosity of the porous membrane. The porous membranesample to be evaluated was sliced by a microtome (product name FCS, madeby Reichert, Austria) to produce a sample for cross-sectionalobservation, the surface of the sample for cross-sectional observationwas coated with an Os layer at a thickness of 6 nm, and the sample wasobserved with a SEM using an accelerating voltage of 2 kV. The tensileelongation at break of the porous membrane and the stress required for10% elongation thereof were measured using a FUDOH RHEO METER RT-2002D·D(made by Rheotech Corp.)

FIG. 7B illustrates a micrograph of a porous membrane of comparativeexample 1. In comparative example 1, a porous membrane of conventionaltechnology was employed in which the openings were formed by a tracketching process. Note that the average opening diameter of the openingsin the porous membrane of comparative example 1 was 5.7 μm, the openingcoverage ratio of the openings was 12.4%, the membrane thickness of theporous membrane was 10.6 μm, the variation coefficient of the openingdiameters of the openings was 35%, the porosity of the porous membranewas 15%, the tensile elongation at break was 150%, and the stressrequired for 10% elongation was 5800 gf/mm².

A porous membrane is prepared with medical paper affixed to both sidesthereof. The medical paper on one face of the porous membrane is removedusing tweezers, and the face from which the medical paper has beenremoved is set face down on a lower channel member. The porous membraneis then soaked in ethanol using a swab to join the porous membrane andthe lower channel member together.

Next, the medical paper on the other face of the porous membrane isremoved using tweezers, and the upper channel member is stacked on theother face of the porous membrane. The positions of the upper channelmember and the lower channel member are aligned while being checking amicroscope, and the upper channel member and the lower channel memberare joined together. Thereby, the blood vessel model of example 1 andthe blood vessel model of the comparative example 1 were respectivelyproduced.

Note that in example 1 and comparative example 1, in order to evaluatethe permeability of the porous membranes to red blood cells, the porousmembranes employed did not have an vascular endothelial cell layer 36nor a cell layer of cells selected from the group consisting of smoothmuscle cells, mesenchymal stem cells, pericytes, and fibroblast cellsprovided on principal faces thereof.

A blood dilution was run through the upper microchannels of the producedblood vessel models and a physiological saline solution was run throughthe lower microchannels thereof. The rate of fluid delivery of the blooddilution and the physiological saline solution was set to 500 μL/min,the internal pressure of the upper microchannel was set to approximately8.7 kPa, and the internal pressure of the lower microchannel was set toapproximately 1.3 kPa so as to establish parameters close to the bloodflow and blood pressure conditions inside actual blood vessels.

Counts of the number of red blood cells inside the lower microchannel,namely, inside the physiological saline solution, after a certain amountof time had elapsed since starting fluid delivery gave a number of redblood cells of 9.2×10⁴ cells/ml in example 1, and a number of red bloodcells of 2.2×10⁴ cells/ml in the blood vessel model of Comparativeexample 1.

This test was able to confirm that the porous membranes of example 1 andcomparative example 1 both had permeability to red blood cells underconditions equivalent to those of blood pressure. Further, in comparisonto the porous membrane of comparative example 1, the porous membrane ofexample 1 was more readily permeable to red blood cells, enablingconfirmation that the porous membrane of the present exemplaryembodiment enables the movement of red blood cells to be suppressed frombeing obstructed.

What is claimed is:
 1. A blood vessel model comprising: a pair ofchannel members, mutually opposing each other, each of which includes anopposing face in which a respective microchannel is formed; and a porousmembrane that includes a plurality of through-holes penetrating in athickness direction, that is disposed between the opposing faces of thepair of channel members, and that partitions between the microchannels,wherein the porous membrane is provided with a vascular endothelial celllayer so as to cover one face facing one of the microchannels, anaverage opening diameter of the through-holes is from 1 μm to 20 μm, andan opening coverage ratio of the through-holes is from 30% to 70%. 2.The blood vessel model of claim 1, wherein a membrane thickness of theporous membrane is less than or equal to half of the average openingdiameter of the through-holes.
 3. The blood vessel model of claim 1,further comprising communication holes, that place the through-holes incommunication with each other, formed inside the porous membrane,wherein: the through-holes are arranged in a honeycomb pattern; avariation coefficient of opening diameters of the through-holes is lessthan or equal to 10%; and a porosity of the porous membrane is greaterthan or equal to 50%.
 4. The blood vessel model of claim 1, furthercomprising a cell layer of cells selected from the group consisting ofsmooth muscle cells, mesenchymal stem cells, pericytes, and fibroblastcells, provided at the other face of the porous membrane facing theother microchannel.
 5. The blood vessel model of claim 1, wherein: atensile elongation at break of the porous membrane is greater than orequal to 50%; and a stress required for 10% elongation of the porousmembrane is less than or equal to 1000 gf/mm².
 6. The blood vessel modelof claim 1, wherein the through-holes have flattened shapes in plan viewincluding a major axis and a minor axis.
 7. The blood vessel model ofclaim 1, wherein the porous membrane includes a porous region in whichthe through-holes are formed, and a non-porous region in which thethrough-holes are not formed.
 8. A method for executing an extravasationtest using a blood dilution containing a drug, the method comprising:providing the blood vessel model of claim 1; running the blood dilutioncontaining the drug in a microchannel that faces the face of the porousmembrane to which the vascular endothelial cell layer is provided; andcounting a number of red blood cells that leak out into a microchannelthat faces the other face of the porous membrane.